Abstract 18201: Src-Dependent Caveolar Trafficking Defects in Hereditary Pulmonary Arterial Hypertension
Patients with hereditary pulmonary arterial hypertension (HPAH) inherit heterozygous germ line mutations in the BMP type 2-receptor, BMPR2, but it is not known how these mutations cause the disease. Bmpr2 mutant mice have endothelial dysfunction and increased vascular permeability, and are more susceptible to the development of pulmonary hypertension. In these studies we explore the mechanisms mediating endothelial barrier dysfunction in Bmpr2+/- mice. Conditionally immortalized pulmonary endothelial cells (ciPECs) from Bmpr2+/- mice have increased permeability to 70kD dextran and albumin, but not to 4kD dextran, suggesting that Bmpr2+/- ciPECs have intact cell-cell junctions but increased transcellular (vesicle mediated) permeability. Caveolae are the predominant vesicle involved in transcellular permeability in endothelium. Transmission EM shows an increase in Caveolar structures in ciPECs and intact endothelium of Bmpr2+/- lungs. Quantitative analysis indicates that Caveolin-1 (Cav1) and Cavin have altered localization from the plasma membrane (PM) in wild type ciPECs to an intracellular localization in Bmpr2+/- ciPECs. In addition, inhibition of Caveolar endocytosis with the Dynamin-2 inhibitor, Dynasore, restores Cav1 localization to the PM in Bmpr2+/- PECs, suggesting there is increased Dynamin-dependent Caveolar endocytosis and trafficking in Bmpr2+/- ciPECs. Bmpr2+/- ciPECs also have increased levels of phosphorylated (pY14) Cav1 associated with increased expression of active Src-kinase (pY416), the kinase responsible for phosphorylating Cav1 and initiating Caveolar endocytosis. The selective Src-family kinase inhibitor PP2, reduces pY14-Cav1 and rescues Cav1 and Cavin localization to the PM in Bmpr2+/- ciPECs. Furthermore, PP2 restores endothelial barrier function in Bmpr2+/- ciPECs demonstrated by a reduction in albumin and 70kD dextran permeability. These data demonstrate a defect in Caveolar trafficking mediated by constitutive activation of Src kinase in Bmpr2+/- ciPECs, and suggest Src kinase inhibitors may ameliorate endothelial barrier dysfunction in HPAH patients.
- © 2013 by American Heart Association, Inc.